A long standing goal of molecular biotechnology has been the ability to design and generate DNA binding proteins that specifically bind at a DNA sequence of choice, rather than rely on the limited set of DNA sequences bound by those proteins identified from nature. To this end, the structures of a number of DNA binding proteins complexed with their DNA target sequence have been determined by crystallography (Lukacs, et al. Nat. Struct. Biol. 7: 134-140 (2000) and the amino acid residues conferring specific DNA base recognition have been determined (Pingoud, et al. Nucleic Acids Res. 29:3705-3727 (2001)). However, to date, rational design experiments in which specific amino acid residues are altered to form DNA binding proteins having new, predetermined specificities have been unsuccessful. For example, attempts to generate restriction endonucleases with new DNA recognition specificities have not achieved their desired goals. As a result, methods have been designed that depend on random alteration of a DNA binding protein, followed by a selection from the pool of randomly altered proteins for those proteins that may bind a differing DNA sequence. Often such attempts result in proteins that bind a relaxed specificity relative to the starting protein or have lowered specificity toward their target DNA binding sequence as compared with similar, non-target DNA sequences.
Nonetheless, an effective method of rational design of binding proteins would permit the expansion of the number of unique recognition sequences that could be bound and acted upon to generate a biological event.